The invention relates to a method of introducing exogenous genetic materials into lymphocytes, in vitro and in vivo, and its use in studying the function and regulation of immune system. Specifically, lymphocytes are genetically engineered to express a truncated Coxsackievirus and/or Adenovirus Receptor (tCAR) protein to allow improved adenovirus vector transfection.
The inefficiency of gene delivery into lymphocytes has hampered the investigation of the pathways that control lymphocyte physiology. Although retrovirus vectors have been used successfully to transduce multiple hematopoietic cell types, experiments involving retroviral transduction of lymphocytes are limited by the difficulty in transducing the entire population of cells, the requirement that the cells be proliferating for viral integration, and the time required for the transduction and expression of the introduced gene. Other approaches to manipulate gene expression in lymphocyte cell lines often require either the generation of cell lines that express the desired gene product, sometimes under the control of an inducible promoter, or the transient transfection of the gene of interest into a fraction of the cells. In addition, the manipulation of gene expression in vivo usually entails the creation of transgenic or gene disrupted mice. These technologies, however, are costly and time consuming, thus have been applied to a few limited cases. Furthermore, the expression of an exogenous gene in transgenic mice is often detrimental to the normal development. The inability to introduce genes easily and efficiently into lymphocytes and examine the consequences of such expression soon after gene delivery limits the characterization of lymphocyte pathways that control cell growth, differentiation and death.
Adenoviral vectors are attractive in that either proliferating or quiescent cells can be transduced, the expression of the introduced gene is evident by as early as 5 hrs after transfection, the vectors can accommodate large insert sizes (7-9 kilobases), and high titer stocks are easily generated [Nevins, J. R. et al., (1997) Meth. Enzymol 283:205-219]. A particular advantage of the adenovirus vectors lies in their ability to transduce the entire cell population. Group C adenovirus (e.g. Ad2 and Ad5) infection requires the high affinity attachment of the viral fiber capsid protein to a cellular receptor and viral penton base binding to certain cellular integrins, followed by cell entry via receptor-mediated endocytosis [Wickham, T. J., et al., (1993) Cell 73:309-319]. Unfortunately, although adenovirus can infect a wide range of cell types, lymphocytes are not very susceptible to adenovirus infection, apparently as a result of the failure of adenovirus to efficiently bind the cell surface and be internalized [DeMatteo, R. P. et al., (1997) Ann. of Surgery 222:229-242; Neering, S. J. et al., (1996) Blood 88:1147-1155; Chu, Y. et al., (1992) Virology 188:793-800]. In part, the inability of adenovirus to enter T cells is due to the very low levels of cellular fiber receptor expressed on these cells [Huang, S. et al., (1997) Journal of Virology 69:2257-2263; Huang, S. et al., (1996) J. Virol. 70:4502-4508]. In addition, T cells express limiting levels of .alpha.V containing integrins, and mitogen-mediated upregulation of .alpha.V integrin expression confers limited infection by adenovirus vectors [Huang et al., (1997) supra].
The cDNA for the cellular receptor for the adenoviral fiber protein, CAR (for Coxsackievirus and/or Adenovirus Receptor), was recently cloned, and the expression of CAR in Chinese hamster ovary (CHO) cells increased their susceptibility to adenovirus infection approximately 100 fold [Bergelson, J. M. et al., (1997) Science 275:1320-1323].
There has been a long-felt need in the art for an efficient gene transfer method for lymphocytes. The invention described herein provides in vitro cell culture systems and in vivo animal models allowing efficient adenovirus-mediated transduction, and subsequently, enabling the art to study diverse functions of lymphocytes as well as to test agents to modulate lymphocyte activity.